Many large proteins are composed of a number of discrete domains, or subunits. Since domain-domain interaction is often involved in function, it is important to understand these interactions. A new thermodynamic model for multidomain proteins, based on results from differential scanning calorimetry (DSC), has been developed and will be tested to determine its usefullness. It is detailed enough to quantify domain-domain interactions using a pairwise interaction term, delta GAB, but simple enough so it can be applied to real systems to obtain definite (albeit approximate) answers regarding the strength of domain interactions and their role in carrying out function. Although estimates of the absolute value of delta GAB can be made in certain instances, the model is particularly suited for obtaining estimates of changes which occur in domain interactions when ligands are bound, mutations are introduced, or when other variables are changed. We plan to investigate, using the model when appropriate, two systems where domain interactions are thought to play a critical role in function: 1.) A well-defined, two-domain, soluble enzyme from yeast- 3-phosphoglycerate kinase (PG32, and 2.) A membrane- spanning protein, the aspartate receptor (AR) of S. typhimurium, a paradigm for receptor/transducer proteins in signalling systems. For each system. DSC results will be obtained to determine the number of domains, identify each, domain in terms of its ligand- binding properties and function, estimate the extent of interaction between domain-pairs, and how this changes with ligand binding, mutations, etc. The DSC results will be supplemented by the direct measurement of ligand-binding constants using rapid, ultrasensitive titration calorimetry. The complete characterization of binding parameters (i.e., n, delta H, delta S and K) will be carried out for single ligands (MgATP) and 3-phosphoglycerate for PGK: stimulus ligands (aspartic acid, cobalt ions) and second messenger components (methyltransferase) for the AR) and for coupled binding of ligand-pairs whose sites are located on different domains.